Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis

Carbohydrate Research 337 (2002) 105–120

www.elsevier.com/locate/carres

Synthesis and biological evaluation of trehalose analogs as

potential inhibitors of mycobacterial cell wall biosynthesis

Jerry D. Rose,^aJoseph A. Maddry,^aRobert N. Comber,^bWilliam J. Suling,^a

Larry N. Wilson,^aRobert C. Reynolds^a,*

^aSouthern Research Institute, Drug Disco6ery Di6ision, 2000 Ninth A6enue South, PO Box 55305, Birmingham, AL 35255-5305, USA

^bCroda Inc., 180 Northﬁeld A6enue, Edison, NJ 08837, USA

Received 5 April 2001; accepted 4 September 2001

Abstract

Analogs of trehalose are reported that were designed to interfere with mycolylation pathways in the mycobacterial cell wall.

Several derivatives of 6,6%-dideoxytrehalose, including N,N%-dialkylamino and 6,6%-bis(sulfonamido) analogs, were prepared and

evaluated for antimycobacterial activity against Mycobacterium tuberculosis H₃₇Ra and a panel of clinical isolates of Mycobac-

terium a6ium. 6,6%-Diaminotrehalose and its diazido precursor were both inactive, but signiﬁcant activity apparently related to

Keywords: Trehalose mono- and di-mycolate; Mycolylation; Antitubercular agents; Antigen 85 complex

valent forces.¹⁷The mycolic acids are a unique group of

functionalized, very long chain (\60 carbon atoms),

a-branched, b-hydroxylated carboxylic acids that are

esteriﬁed to terminal arabinan branches of this polysac-

charide, thus forming the core of the lipid envelope.¹⁸

Since one of the more effective antituberculosis drugs,

isoniazid, acts through inhibition of mycolic acid bio-

genesis,¹⁹and since the lipid layer itself is thought to

hinder uptake of other potential drugs, disruption of

the mycolate structure is a compelling strategy for the

development of novel agents.

Trehalose is vitally important to the mycolate econ-

omy of mycobacteria.²⁰Both 6-mycolyltrehalose and

6,6%-dimycolyltrehalose are believed to act as donors for

mycolate transfer to arabinan during envelope elabora-

tion. Recently, it was reported²¹that the antigen 85

complex (ag85 A, B, and C) appears to play a role in

cell-wall biogenesis, and, in particular these proteins

possess a mycolyltransferase capability that utilizes tre-

halose mono- and di-mycolates. Our laboratories have

synthesized a series of trehalose derivatives designed to

inhibit this mycolyltransferase activity. In the design of

these agents, we reasoned that for transesteriﬁcation to

proceed efﬁciently there would likely be an activating

1. Introduction

Tuberculosis, although nominally a curable disease,

remains one of the primary health threats worldwide.^1,2

The appearance of drug-resistant, particularly multiple

drug-resistant, forms of the disease throughout the

world has lead to the realization that tuberculosis may

again become an incurable disease.^3–10In fact, for some

multiple drug-resistant strains, there are now few treat-

ment options.¹¹World health ofﬁcials are calling for a

concerted effort to understand the biochemistry and

pathology of the bacterium that may lead to the devel-

opment of new, more effective, and safer vaccines and

drugs.^12,13In particular, new drugs with novel mecha-

nisms of action will be required to treat drug-resistant

forms of tuberculosis.^14–16

One of the chief distinguishing characteristics of my-

cobacteria is a thick lipid envelope, the various con-

stituents of which are bound to an arabinogalactan

polysaccharide coat through both covalent and non-co-

* Corresponding author. Fax: +1-205-5812877.

E-mail address: reynolds@sri.org (R.C. Reynolds).

PII: S0008-6215(01)00288-9

106

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

Scheme 1. Reagents and conditions: (a) C₆H₅CHO, ZnCl₂, rt, 6 days; (b) C₆H₅COCl, pyridine, rt, 64 h; (c) NBS, K₂CO₃, CCl₄,

reﬂux, 2 h; (d) NaN₃, CH₃OH, rt, 48 h; (e) NaOCH₃, CH₃OH, rt, 48 h, cation resin exchange H⁺form; (f) H₂, 5% Pd–C, MeOH,

28 h; (g) H₂, Pd–C, EtOH, HCl; (h) NaHCO₃–H₂O or g without additon of HCl.

moiety, perhaps an aspartate or glutamate residue and/

or possibly a basic function such as a histidine that

could catalyze the reaction through a general acid–base

mechanism. This activation is probable irrespective of

whether an enzyme-bound intermediate is present.

Though the precise mechanism of mycolyl transfer was

not known at the inception of our program, a recent

trehalose derivatives: sulfonamides, alkylamines, amidi-

nes, and guanidine, and to use biological assay for

antimycobacterial activity to guide further synthetic

efforts. 6,6%-Diamino-6,6%-dideoxytrehalose (7), a poten-

tially useful starting material, has been obtained

previously²³by tosylation of anhydrous a,a-trehalose

followed by direct displacement with ammonia, or in

three steps by the sequence 6,6%-ditosylate6,6%-di-

azido6,6%-diamine. However, in a single pilot experi-

ment, tosylation gave a complex mixture of products

that required tedious column separation and gave a low

yield of the desired component, a result that has been

conﬁrmed by others.^24,25

A better route that allowed synthesis of 7 in multi-

gram quantities is outlined in Scheme 1. It uses well-

documented methodology and, with trivial variations, is

adaptable to either the benzyl- or benzoyl-protected

sugar. In addition, Scheme 1 afforded the 6,6%-diazido

compound 6, which we wanted for bioassay, as well as

compounds 4 and 8, both of which saw limited utility

as starting materials for some of the target compounds

in Table 1. Anhydrous a,a-trehalose (1) was obtained

from the commercially available dihydrate either by

prolonged drying in vacuo at 78 °C²⁶or by azeotropic

distillation with pyridine.²⁷Treatment of 1 with freshly

distilled benzaldehyde–anhydrous ZnCl₂followed by

perbenzoylation gave 2²⁶and 3^28–30in yields of 98 and

80%, respectively. The conversion of 3 into 4 in Scheme

1 followed essentially the procedures given by Hanes-

sian.²⁹The only signiﬁcant departure was the substitu-

tion of anhydrous K₂CO₃for BaCO₃in step (c) leading

to 4. Also, isopropyl alcohol was substituted for ace-

tone–ether–pentane as recrystallization solvent. Both

methods gave ꢀ80% puriﬁed yield of 4. Since adequate

publication²¹of the crystal structure of ag85C at 1.5 A

,

reveals a catalytic triad formed by Ser 124, Glu 228,

and His 260, reinforcing our original hypothesis. Con-

sequently, analogs with the carbonyl replaced by a

complementary basic group (amine or amidine) that

could interact with an acidic function through a salt

bridge were targeted. Furthermore, acidic sulfonamide

analogs that could take advantage of a basic function

within the active site were also prepared. Finally, a

hydrophobic group that could partially occupy the

mycolate binding pocket was considered essential, since

it is well known that hydrophobic forces typically

provide most of the ligand binding energy.²²

Several derivatives of 6,6%-diamino-6,6%-dideoxytre-

halose (7), including N,N%-dialkylamino (compounds

12, 15a–d, 18a–c, and 21) and 6,6%-bis(sulfonamido)

(compounds 11a–d), were prepared and evaluated for

antimycobacterial activity against Mycobacterium tu-

berculosis H₃₇Ra and a panel of clinical isolates of

Mycobacterium a6ium.

2. Results and discussion

Preparation of key intermediates.—Our initial syn-

thetic goal was to prepare representatives of several

classes of 6,6%-bis(substituted-amino)-6,6%-dideoxy-a,a-

Table 1

No.

SRI no.

R¹and R²

MIC (mg/mL) ^a

MTB

MAV

H37Ra

NJ168

NJ211

NJ1854 NJ3009 NJ3350 NJ3404

6

7

8633

8998

8706

8667

8648

8649

8855

8792

8793

8982

8980

9009

9029

8979

9046

8849

R¹=R²=N₃

ND ^b

ND

\128

16–32

\32

ND

\128

4

\128

32–64

\64

\128

8

ND

\128

64

\32

ND

\128

2

ND

32–64

ND

\128

ND

32–64

ND

\128

2

\128

ND

16–32

\64

\128

ND

\128

32–64

\64

\128

4

R¹=R²=NH₂

/

11a

11b

11c

11d

12

15a

15b

15c

15d

18a

18b

18c

21

R¹=R²=CH₃(CH₂)₃SO₂NHꢀ

R¹=R²=CH₃(CH₂)₇SO₂NHꢀ

R¹=R²=CH₃(CH₂)₁₅SO₂NHꢀ

R¹=R²=C₆H₅SO₂NHꢀ

R¹=R²=[CH₃(CH₂)₁₁]₂Nꢀ

R¹=R²=CH₃(CH₂)₇NHꢀ

R¹=R²=CH₃(CH₂)₁₁NHꢀ

8

R¹=R²=CH₃(CH₂)₁₇N(CH₃)ꢀ

R¹=R²=[CH₃(CH₂)₃CH(Et)CH₂]₂Nꢀ

R¹=R²=CH₃(CH₂)₅NHꢀ

\128

128

\128

128

\128

8

128

ND

\128

64

\128

\1.3513

64

3

\128

ND

(

ND

R¹=R²=CH₃(CH₂)₉NHꢀ

\1.3513 \1.3513 \1.3513 ND

32

\128

R¹=R²=[CH₃(CH₂)₃CH(Et)CH₂NHꢀ

R¹=CH₃(CH₂)₃CH(Et)CH₂NHꢀ; R²=OH

R¹=R²=CH₃(CH₂)₄C(NH)NHꢀ, mixture with ꢀ10%

[R¹=CH₃(CH₂)₄C(NH)NHꢀ and R²=CH₃(CH₂)₄CONHꢀ]

R¹=NH₂; R²=CH₃(CH₂)₁₀C(NH)NHꢀ

R¹=R²=CH₃(CH₂)₁₀C(NH)NHꢀ

R¹=R²=H₂NC(NH)NHꢀ

64

\128

16

\128

32

ND

105

\128

120

24,25

ND

26

27

32

8984

8983

9026

ND

\128

16

\128

ND

\128

ND

8

\128

EMB ^c

AMK ^c

8

16–32

1–2

8

1–2

8–16

0.5–1

32

2–4

16

1–2

0.5–2

^aThe minimum inhibitory concentration (MIC) was determined for M. tuberculosis (MTB H37Ra) and several M. a6ium (MAV) clinical isolates using either a

macrodilution broth assay or a colorimetric microdilution broth assay.

^bND, not done.

^cEthambutol (EMB) and amikacin sulfate (AMK) were used as positive controls. MICs are given as the range of values obtained with multiple assays.

108

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

Scheme 2.

procedures for the conversion of 1 to 4 have already

been referenced, experimental details have not been

repeated, but some departures from the literature²⁹in

the sequence leading from 4 to 7 justify inclusion of

compounds 5–7 in Section 3.

the presence of excess Et₃N gave intermediates 10a–d

in moderate to good yields as brittle foams. Although

the migration product 9 was present in the crude reac-

tion mixtures, it was easily removed by silica gel

column chromatography. Conventional deprotection

with NaOMe–MeOH gave target compounds 11a–d as

crystalline solids.

Starting material 7 was found to be poorly soluble in

a variety of common reaction solvents, including pyri-

dine and N,N-dimethylformamide. This problem was

relieved somewhat by the synthesis of compound 8,

obtained easily by catalytic hydrogenation of diazide 5

in the presence of hydrochloric acid to protonate the

amine as it formed. This modiﬁcation was required to

block the well-known oxygen-to-nitrogen benzoyl mi-

gration. A similar acyl migration in the trehalose sys-

tem has been reported.³¹The problem of benzoyl

migration in precursor 8 could have been avoided by

use of the corresponding hexa-O-benzyl compound,³¹

however, we employed intermediates 4 and 8 as precur-

sors in early experiments simply because they were on

hand. In a case where migration was allowed to pro-

ceed in order to obtain a sample of 9 for characteriza-

tion, it was observed that in CH₂Cl₂the process was

fairly slow at room temperature, with several days

being required for completion. Also, the migration is

not a process of random benzoate ester aminolysis,

N-Alkylamines.—Several different approaches were

used to prepare a series of N-alkylated derivatives of

diaminotrehalose (Fig. 1). An attempt to directly alky-

late 7 with dodecyl iodide in dilute N,N-dimethylac-

etamide (DMAc) solution at room temperature showed

insigniﬁcant product formation after 3 days. Increasing

the temperature to 50 °C gave a complex mixture of

N-alkylated, mostly quaternized, products. None of the

desired 6,6%-bis(monoalkylamine) could be isolated, but

a few milligrams of the 6,6%-bis(dialkylamino) com-

pound 12 was obtained by column chromatography for

bioassay. Repetition of this reaction at room tempera-

ture using the benzoyl-protected compound 8 as start-

ing material showed the same unreactivity. After 3

days, an 80% yield of benzoyl migration product 9 was

recovered. An attempt to condense both amino groups

of 8 with dodecyl aldehyde, followed by reduction of

the expected intermediate 13 (Fig. 1) to the desired

6,6%-didodecyl compound was unsuccessful. Mass-spec-

tral analysis showed several unidentiﬁed components of

high mass (polymeric material?), but none of the ex-

pected 13.

1

since H NMR spectral analysis showed that a single

species, structure 9, was present. Compound 8 could be

stored in a freezer under dry conditions for several

weeks. A far more signiﬁcant limitation of 7 as a

starting material was the unexpectedly weak nucle-

ophilicity of its amino groups, a characteristic shared

by its benzoylated derivative 8. In fact, of the several

classes of target compounds mentioned above, only

sulfonyl chlorides (Scheme 2) were sufﬁciently reactive

with 8 to generate the desired products in acceptable

yields. Attempted aminolysis of a series of methyl imi-

date esters (23, Scheme 2) by 7 gave poor results.

However, it is noteworthy that reaction of 7 with a

masked guanidino precursor (Fig. 3) with elimination

of methanethiol went slowly in N,N-dimethyl-

formamide.

An alternative and more successful approach involv-

ing direct displacement of the 6,6%-dibromotrehalose

with alkylamines was used for the preparation of com-

pounds 15 a–d (Fig. 1). Reaction of the 6,6%-dibro-

motrehalose

4

with n-octylamine and with

n-dodecylamine gave intermediates 14a and 14b, and

prolonged reaction with these primary amines removed

the benzoyl groups as well, thus generating target com-

pounds 15a and 15b in a single step. However, prepara-

tion of intermediates 14c and 14d by reaction of 4 with

secondary amines required activation by NaI and high

reaction temperatures, followed by conventional depro-

tection with NaOMe–MeOH or NH₃–MeOH to gener-

ate 15c and 15d.

It became obvious that a different and more reactive

intermediate than either 4, 7, or 8 was needed. The

benzoyl-protected 6,6%-ditriﬂate 16 (Fig. 1) seemed at-

tractive, as the acetyl-,³²benzyl-,³²and benzoyl-

Sulfonamides.—The ﬁrst group of potential antimy-

cobacterial agents are the sulfonamido compounds

shown in Scheme 2. The free base form of 8 was

generated in situ from 8·2HCl with NaHCO₃. Reaction

of 8 with four commercially available sulfonyl chlorides

(butyl, octyl, hexadecyl, phenyl) in CH₂Cl₂solution in

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

109

Fig. 1. N-Alkylated derivatives of diaminotrehalose.

protected³³ditriﬂates have been reported. In addition to

these literature procedures, we had published³⁴com-

plete experimental details for conversion of anhydrous

trehalose to 16 in four steps. As expected, the reactions

of 16 with alkylamines to give compounds 17a–c were

faster and cleaner than previous methods, and in retro-

spect, all of the alkylamines reported in Fig. 1 could

have been prepared most easily from 16. An unexpected

result was encountered in the cases where conventional

NaOMe in MeOH debenzoylation was employed to

obtain the target alkylamines 14 a–d and 17 a–c. All

benzoyl-protected starting materials (14c,d; 17b,c) were

consumed within 1 h to give the expected product as an

iodine-absorbing, but non-UV-active (ﬂuorescence

quenching) spot on TLC, accompanied by a slightly

faster-moving, UV-active spot that was not signiﬁcantly

affected by increasing reaction time to 3 h. However,

exposure of crude 18b to liquid NH₃for 24 h dimin-

ished the byproduct spot and increased the desired

product spot signiﬁcantly. This extraordinary base sta-

bility plus MS evidence that showed 18b with one

remaining Bz group suggested that the byproduct was

an N-alkyl-N-benzoyl compound. This was conﬁrmed

in the preparation of 18c by isolation of the byproduct.

MS showed 18c with one extra Bz group (m/z 669), and

IR showed no benzoyl ester (CꢁO) absorption, but was

consistent with a substituted benzamide. Careful rein-

spection of the starting material 17c (NMR) showed

that O to N benzoyl migration had not already oc-

curred; thus, the N-benzoyl byproduct arose during the

deblocking procedure. All of these crude deprotection

products could have been reworked via the liquid NH₃

method to maximize yields, but our main emphasis was

simply to obtain an adequate quantity of pure target

compounds for biological assay.

We had reported previously (Ref. 34; page 175) that

2,3,4,2%,3%,4%,6%-hepta-O-benzoyltrehalose had been iso-

lated as a byproduct in the synthesis leading to 16.

Reaction of this compound with triﬂic anhydride gave

the monotriﬂate 19. Displacement with 2-ethylhexy-

lamine followed by debenzoylation gave target com-

pound 21 (Fig. 1) in which only one glucopyranose ring

bears an alkylamine substituent.

Amidines.—Another class of potential antimycobac-

terial agent that received considerable effort but limited

success is illustrated by compounds like 24 shown in

Fig. 2. Following a literature procedure,³⁵a series of

methyl imidate esters 23a–d (Fig. 2) were prepared by

the dry HCl-catalyzed addition of anhydrous MeOH to

the cyano group of four commercially available nitriles,

[RCN 22a–d, where R=C₅H₁₁(22a), C₁₁H₂₃(22b),

C₆H₅CH₂(22c), (2-naphthyl)CH₂(22d)]. The products

23a–d were obtained as solid hydrochloride salts with

limited solubility in cold Et₂O, which was used to wash

out two byproducts (namely the corresponding amide,

RCONH₂, and the ester, RCO₂Me). Except for an

NMR spectrum for 23d, which was obtained to conﬁrm

Fig. 2. Amidine derivatives of trehalose.

110

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

its structure, these intermediates were used without

characterization. Mass-spectral data, however, obtained

on subsequent reaction products known to contain the

appropriate unreacted 23 provided additional conﬁrma-

tion. Reaction of 6,6%-diamino-6,6%-dideoxytrehalose (7)

with an excess of each of these imidate esters (free base)

was expected to effect aminolysis of the esters to gener-

ate trehalose derivatives substituted at the 6-positions

by imidoamide (amidine) groups to give target struc-

tures 24, 27, 28, and 29. Unfortunately, the weak

nucleophilicity of the amino groups in 7 was very

evident here. For the two aliphatic esters (23a and 23b),

formation of the mono(amidine) by reaction at only

one 6-position could be detected by TLC after a few

hours, increasing to perhaps 25–30% after a week or

more. There was even less of the 6,6%-bis(amidine),

probably no more than 10–15% of the reaction product

as estimated by TLC. For the two esters with aromatic

substituents (23c and 23d), no amidine products (28 or

29 or their mono(amidine) analogs) were ever isolated

in sufﬁcient quantity and purity to justify analysis and

bioassay. A solution of 7 and 23d in methanol, where

R=(2-naphthyl)CH₂ꢀ was also reﬂuxed under N₂for

20 h, but no product 29 was obtained. In a parallel

halose components applied in water solution was read-

ily adsorbed, but a very large volume of eluant

containing 50% MeOH was required to elute the or-

ganic components, along with a large quantity of plas-

tic material leached from the column. The net effect

was to substitute one group of contaminants for

another.

Because of these problems, only compounds 24, 26,

and 27 were characterized sufﬁciently to justify biologi-

cal evaluation. Compound 24 appeared to be of high

purity by TLC and MS, but the sample was submitted

for biological evaluation as a mixture of ꢀ90% 24 and

ꢀ10% 25, as their acetic acid salts, based on elemental

analysis and NMR data. Likewise, the mono(amidine)

26 and the corresponding bis(amidine) 27 gave satisfac-

tory mass and ¹H NMR spectra, but were contami-

nated with silica. However, compound 27 showed

signiﬁcant antimycobacterial activity.

Diguanidino.—The 6,6%-diguanidino compound 32

(Fig. 3) was obtained by a route that avoided difﬁcult

puriﬁcation problems like those associated with the

similar

amidines.

N,N%-Bis(benzyloxycarbonyl)-S-

methylisothiourea (30) (Fig. 3) was obtained by react-

ing S-methylisothiourea with benzyl chloroformate

according to a literature procedure.³⁶Reaction of 30

with 6,6%-diamino-6,6%-dideoxytrehalose (7) in DMF for

6 days gave intermediate 31 as a mixture with ꢀ10% of

a similar product with three Cbz groups instead of four

as shown by MS: 827 [M+H]⁺. Deprotection of the

mixture 31 by catalytic hydrogenolysis in MeOH with

10% Pd-on-charcoal in the presence of a little CHCl₃to

serve as an in situ source of HCl³¹gave the 6,6%-

diguanidino compound 32 as its dihydrochloride salt

with no need for further puriﬁcation.

Antimycobacterial acti6ity.—The compounds listed

in Table 1 were evaluated for antimycobacterial activity

against M. tuberculosis strain H₃₇Ra and a panel of

three to ﬁve clinical isolates of M. a6ium. Minimum

inhibitory concentrations (MIC) for each compound

were determined using either a colorimetric microdilu-

tion broth assay (for a detailed description of the

procedures, see Ref. 38) or a macrodiltuion broth as-

say. For ethambutol and amikacin, used against the

same strains as controls, MIC’s ranged from 8 to 32

mg/mL and 0.5 to 4 mg/mL, respectively.

series of reactions of methyl-5-amino-5-deoxy-D-

arabinopyranoside with ﬁve imidate esters [23a–d plus

a ﬁfth with R=CH₃(CH₂)₁₄ꢀ], only reactions with 23b

and 23d gave sufﬁcient arabinose-5-(amidine) for

bioassay (Dr Joyce Friedrich, unpublished results).

Besides sluggish reactivity, another troublesome as-

pect of the amidine chemistry was the nearly intractable

puriﬁcation problems. A typical reaction mixture con-

tained large quantities of unreacted imidoester, along

with its byproducts RCONH₂and RCO₂CH₃. Tre-

halose components included unreacted diaminotre-

halose, the 6-mono(amidine), the 6,6%-bis(amidine), and

their corresponding normal amides formed either by

degradation of the amidines or by aminolysis of the

minor ester contaminant present in the starting material

23. Chromatographic resolution of this latter group was

hindered by close spacing of bands and by their highly

polar nature, which led to severe trailing on the

column. No neutral, polar solvent, or solvent mixture

was found that produced adequate mobility and resolu-

tion on either silica gel or cellulose, but the best com-

promise was found to be mixtures containing both

alcohols and acetic acid, such as n-BuOH–HOAc–H₂O

or CHCl₃–MeOH–HOAc, used with coarse silica gel

(70–230 mesh) contained in a ﬂash column pressurized

with N₂to provide an adequate ﬂow rate. The presence

of the acetic acid sharpened the resolution considerably

while at the same time leaching silica from the column

packing and accelerating the otherwise slow degrada-

tion of amidine to normal amide. In a single trial

puriﬁcation with styrene–divinylbenzene copolymer

beads (Bio-Beads™ SM-4), a complex mixture of tre-

Fig. 3. 6,6%-Diguanidino trehalose derivatives.

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

111

6,6%-Diamino-6,6%-dideoxytrehalose (7) and its di-

azido precursor 6 were both inactive, but signiﬁcant

activity apparently related to aliphatic chain length was

found among the sulfonamides, N-alkylamines, and

one of the amidines. Many more examples of each class

would be required to fully elucidate structure–activity

relationships, but among the compounds tested, the

following trends were noted.

The C₈-alkylsulfonamido compound 11b was the

most active compound of this group; the C₁₆, C₄and

phenyl analogs were inactive at the highest concen-

trations tested.

Among the N-alkylamines, the C₈, C₁₀, and C₁₂

compounds 15a, 18b, and 15b showed consistently

good activity against the mycobacterial panel; the C₆

analog 18a and the branched-C₈(2-ethylhexyl) com-

pound 18c showed a broader range of activities

against different strains of mycobacteria. 6,6%-Bis(ter-

tiaryamino) compounds 12 and 15d were less active

than their bis(secondary-amino) counterparts 15b

and 18c. Tertiary amine 15c with a C₁₈substituent

was also inactive. It was interesting that compound

21, substituted in only one glucopyranose ring with

the (2-ethylhexyl)amino group, was inactive, but the

6,6%-disubstituted counterpart 18c was moderately

active.

Molecular Spectroscopy Section of the Southern Re-

search Institute or by Atlantic Microlab, Inc., Nor-

cross, GA. Where solvents are included in the reported

1

analysis, their presence was conﬁrmed by the H NMR

spectrum. Analtech precoated (250 mm) Silica Gel G

(F) plates were used for thin-layer chromatography

(TLC), and spots were detected by irradiation with a

UV-254 light, by absorption of iodine vapor, or by

charring after a (NH₄)₂SO₄–H₂SO₄spray. Column

chromatography in either the ﬂash column or gravity

mode was performed with 230–400 mesh silica gel (SG)

using the slurry method of column packing, except in a

few cases where the coarser 70–230 mesh is speciﬁed.

Mass spectra were recorded on a Varian/MAT 311A

double-focusing mass spectrometer in the fast-atom

bombardment (FAB) mode. Addition of a trace of LiCl

to some samples gave an [M+Li]⁺cluster peak that

was stronger than the [M+H]⁺peak. For many com-

pounds a particularly prominent mass peak was gener-

ated by rupture on both sides of the glycosidic bond to

give two glucopyranosyl units. In the mass spectral data

for each compound the symbol 1/2[M−16]⁺should be

understood to represent the appropriate glucopyranosyl

unit complete with its various substituent groups. In-

frared spectra were recorded on a Nicolet FT IR spec-

trophotometer, model 10DX, using pressed KBr discs

or capillary ﬁlms. Peak positions are reported in

The C₁₂bis(amidine) 27 showed good activity

against the two M. a6ium strains tested, but again,

the monosubstituted counterpart 26 was inactive for

the single strain tested. The C₆bis(amidine) 24 was

inactive.

The 6,6%-diguanidino compound 32 was inactive.

In addition to the compounds in Table 1, the ben-

zoyl-protected precursors 10b, 10c, 10d, 17a, and 17b

were also screened; all were inactive.

wavenumbers (w) in cm⁻^{1 1}H NMR spectra were

.

recorded on a Nicolet/GE NT300NB spectrometer op-

erating at 300.635 MHz. Chemical shifts (l) are re-

ported in ppm downﬁeld from internal Me₄Si. Chemical

shifts of multiplets are measured from the approximate

center of the multiplet, and coupling constants (J) are

reported in Hz. Because of the remarkable symmetry of

the trehalose molecule, equivalent protons in both rings

have the same chemical shift, but it should be noted

that the CH₂protons located at the 6-position in both

rings are nonequivalent, and they may couple with

other protons with only slightly different J values,

giving rise to apparent triplets or quartets on ﬁrst-order

3. Experimental

General methods.—Melting points were determined

by the capillary method on a Mel-Temp apparatus and

are uncorrected. Reaction temperatures below the boil-

ing point of the indicated solvent were external oil bath

temperatures. Evaporations were carried out in vacuo

on a rotary evaporator or under high vacuum by

short-path distillation into a glass trap cooled in dry

ice–acetone. Samples were dried under high vacuum

over P₂O₅at rt unless an elevated temperature is spe-

ciﬁed. Many of the alkylamines reported herein were

obtained as soft, waxy, but tractable, foams with low,

indistinct melting points by ﬂash evaporation from

concentrated solution in CH₂Cl₂. Those with the longer

alkyl chains exhibited detergent behavior in water–or-

ganic solvent mixtures, and some attempts at recrystal-

lization from alcohol solvents resulted in the formation

of gels. Elemental analyses were performed by the

1

inspection which are, in fact, two doublets. In the H

NMR data for the reported compounds of Scheme 1

and for the complete set of protected and deprotected

compounds of Scheme 2, we have identiﬁed and docu-

mented the relevant couplings. However, inspection

shows little variation in coupling patterns and J values

within a structural group; therefore, the other schemes

include detailed coupling data for a representative com-

pound of that group. Similar data for other compounds

are available upon request. UV spectra were not ob-

tained for all samples because of the lack of signiﬁcant

absorption above 200 nm for many of the deprotected

target compounds, but for selected samples they were

determined in 0.1 N HCl (pH 1), pH 7 buffer, and 0.1

N NaOH (pH 13) with a Perkin–Elmer UV–Vis-near

infrared model Lambda 9 spectrophotometer. Maxima

112

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

are reported in the format: u_maxin nanometers (m×

10⁻³).

3.55 (m, 2 H, H-3), 3.96 (m, 2 H, H-5), 4.91 (m, 6 H,

H-1, OH-2, and OH-3 overlap), 5.13 (d, 2 H, J 4.8 Hz

OH-4). A trace of 2-PrOH was noted. Anal. Calcd for

C₁₂H₂₀N₆O₉·0.1 C₃H₈O: C, 37.09; H, 5.26; N, 21.10.

Found: C, 37.05; H, 5.15; N, 21.15.

6,6%-Diazido-2,3,4,2%,3%,4%-hexa-O-benzoyl-6,6%-di-

deoxy-h,h-trehalose (5).—Intermediates 2–4 (Scheme

1) had been prepared from anhyd a,a-trehalose (1) by

known procedures.^26–30Under an N₂atmosphere, a

stirred mixture of 4 (91.5 g, 83.7 mmol) and excess

NaN₃(71.4 g, 1.1 mol) in dry DMF (1 L) was heated

for 14 h at 65 °C. After cooling, the dark mixture was

poured with vigorous mechanical stirring into 15 L of

ice and water. The precipitate was collected by ﬁltra-

tion, washed with cold water and cold isopropyl alco-

hol, and then recrystallized from 5 L of boiling

isopropyl alcohol with ﬁltration through a preheated

Buchner funnel. The dense crystalline deposit was col-

lected, washed with cold 2-PrOH, and dried to constant

weight at rt; yield 76.5 g (90%); mp 184–185 °C. A

200-mg portion was recrystallized from 2-PrOH (40

mL) to give an analytical sample. The long white

needles were dried at rt overnight and then at 65 °C for

1 h; yield 187 mg; mp 185–186 °C (lit.²⁹180–182 °C).

MS: (with LiCl) m/z 1023 [M+Li]⁺, 500 (1/2[M−

16])⁺. IR: 2107 cm⁻¹(azido), 1730 cm⁻¹(ester CꢁO).

¹H NMR (Me₂SO-d₆): l 3.27 (d, 4 H, J 4.1 Hz, 6-CH₂),

4.02 (m, 2 H, J_4,59.9, J_5,6=J_5,6%=4.1 Hz, H-5), 5.59

(dd, 2 H, J_3,49.6 Hz, H-4), 5.67 (dd, 2 H, J_1,23.5, J_2,3

10.0 Hz, H-2), 5.77 (d, 2 H, H-1), 6.10 (app. t, 2 H,

H-3), 7.41–7.69, 7.83, 8.06 (complex m, 30 H, Bz).

Anal. Calcd for C₅₄H₄₄N₆O₁₅: C, 63.78; H, 4.36; N,

8.26. Found: C, 63.60; H, 4.28; N, 8.05.

6,6%-Diamino-6,6%-dideoxy-h,h-trehalose (7).—A so-

lution of 6 (8.05 g, 20.53 mmol) in CH₃OH (900 mL)

was hydrogenated at atmospheric pressure by stirring

with 5% Pd–C catalyst (1 g) for 27 h. The mixture was

ﬁltered through a Celite pad under N₂pressure, and the

catalyst was washed with 2×100 mL of hot CH₃OH.

Evaporation of the ﬁltrate gave a white solid, 2.93 g

(42%), which was puriﬁed by extraction into CH₃OH

(150 mL) in a Soxhlet apparatus. The solid that de-

posited in the cooled extract was dried at 82 °C for 18

h to give a sample that analyzed as the 0.6 MeOH

solvate. After redrying overnight at 82 °C and 2 h at

100 °C, the composition was virtually unchanged; yield

1.87 g (25%); mp sinters and chars gradually above

ꢀ218 °C. MS: m/z 341 [M+H]⁺, 162 (1/2[M−16]⁺).

¹H NMR (Me₂SO-d₆): l 2.56, 2.60 (2d, 2 H, J_6,6%13.3,

J_5,6%6.4 Hz, 6-CH₂), 2.75, 2.80 (2d, 2 H, J_5,63.3 Hz,

6-CH₂), 3.06 (app. t, 2 H, J_3,48.5, J_4,58.8 Hz, H-4),

3.26 (dd, 2 H, J_1,23.5, J_2,39.6 Hz, H-2), 3.54 (app. t, 2

H, H-3), 3.61 (m, 2 H, H-5), 0.8–2.1 and 4.0–5.8 (very

broad, NH, OH, best seen in integral). MeOH was

noted. Anal. Calcd for C₁₂H₂₄N₂O₉·0.6 CH₃OH: C,

42.09; H, 7.40; N, 7.79. Found: C, 42.33; H, 7.33; N,

7.78.

6,6%-Diamino-2,3,4,2%,3%,4%-hexa-O-benzoyl-6,6%-di-

deoxy-h,h-trehalose dihydrochloride (8).—A suspension

of 5 (5.00 g, 4.92 mmol) in EtOH (1 L) and 1.0 N HCl

(11.0 mL) was hydrogenated at atmospheric pressure by

stirring with 5% Pd–C catalyst for 28 h. After ﬁltration

of the mixture through a thin Celite pad and liberal

washing of the pad with EtOH, the ﬁltrate was evapo-

rated using a lukewarm water bath. The residue was

puriﬁed by reprecipitating twice from MeOH (50 mL)

solution by dropwise addition of Et₂O (250 mL). The

solid was collected by ﬁltration under N₂pressure,

washed with Et₂O, and dried at rt overnight and at

65 °C for 3 h; yield 3.14 g (57%). MS: m/z 965 [M+

6,6%-Diazido-6,6%-dideoxy-h,h-trehalose

(6).—Solid

NaOMe (1.59 g, 30.0 mmol) was added to a suspension

of 5 (30.0 g, 29.5 mmol) in dry MeOH (1500 mL), and

the mixture was stirred at rt for 48 h, having become a

clear solution after ꢀ20 h. The solution was neutral-

ized by portionwise addition of Dowex 50W-X8 (H⁺

form) cation exchange resin. The resin was ﬁltered off,

and evaporation of the ﬁltrate gave a semisolid residue

that was washed by trituration and decantation with

hexane (3×50 mL) to remove methyl benzoate. A

solution of the residual solid in hot isopropyl alcohol

(700 mL) was treated with charcoal (5 g) and Celite (20

g) and ﬁltered through a thin Celite pad to remove a

colloidal turbidity assumed to be NaCl. On cooling, the

clear ﬁltrate deposited dense white crystals, which were

collected, washed with 2-PrOH, and dried, ﬁrst at rt

and then at 82 °C for 18 h; yield 8.41 g (73%); mp

211–212 °C (dec with gas evolution); lit.²⁹mp 195–

198 °C. Concentration of the ﬁltrate to ꢀ100 mL gave

a second crop of lower melting material; 3.0 g (26%);

mp 197–200 °C. Recrystallization of a 200 mg portion

of the ﬁrst crop from 2-PrOH (30 mL) to give a sample

for analysis did not change the melting point; yield 151

mg. MS: m/z 393 [M+H]⁺. IR: 2106 cm⁻¹(azido). ¹H

NMR (Me₂SO-d₆): l 3.10 (complex m, 2 H, H-4), 3.30

(complex m, 2 H, H-2), 3.39 (d, 4 H, J 4.0 Hz, 6-CH₂),

1

H]⁺. H NMR (Me₂SO-d₆): l 2.96, 3.02 (2d, 2 H, J_5,6

7.0, J_6,6%13.6 Hz, 6-CH₂), 3.08, 3.12 (2d, poorly re-

solved, 2 H, J_5,6%2.4 Hz, 6-CH₂), 4.20 (m, 2 H, J_4,510.1,

J_5,6%2.4 Hz), 5.76 (app. t, 2 H, J_3,49.5 Hz, H-4), 5.92,

5.96 (m, 4 H, J_1,23.5, J_2,39.7 Hz, H-1 and H-2), 6.27

(app. t, 2 H, H-3), 7.36–7.80, 8.13 (complex m, 30 H,

Bz), 8.26 (very br, NH₂+H⁺). Very large water peak

noted. Anal. Calcd for C₅₄H₄₈N₂O₁₅·2 HCl·4.3 H₂O: C,

58.15; H, 5.30; N, 2.51. Found: C, 58.18; H, 5.00, N,

2.73.

2,3,2%,3%-Tetra-O-benzoyl-6,6%-bis(benzoylamino)-6,6%-

dideoxy-h,h-trehalose (9).—A byproduct believed to be

compound 9 had been observed to form slowly at rt in

reaction mixtures involving 8 in neutral or basic media.

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

113

An authentic sample of 9 was prepared for positive

identiﬁcation by modifying the procedure for prepara-

tion of 8. The procedure for preparation of 8 from 5

was repeated exactly except that no HCl was added,

and the reaction ﬂask was warmed for 5 h in a 50 °C

water bath during the hydrogenation. After 24 h, ﬁltra-

tion and evaporation of the ﬁltrate gave 4.7 g of crude

product. Column chromatography on silica gel with

97:3 CHCl₃–MeOH as eluant gave three main compo-

nents. The ﬁrst was a stable white solid (2.44 g, 51%)

that was shown by spectral data to be 9. The analytical

sample was obtained by two recrystallizations from

EtOH followed by drying for 6 h at 65 °C; mp 201–

202 °C. MS: m/z 965 [M+H]⁺, 474 (1/2[M−16]⁺),

105 [Bz]⁺. IR: 1731 cm⁻¹(ester CꢁO), 1637 cm⁻¹

satd NaHCO₃, and water. Evaporation of the dried

(Na₂SO₄) organic layer gave 0.75 g of yellow foam.

Puriﬁcation of the crude product by chromatography

on silica gel with (99:1) CHCl₃–MeOH as eluant gave

10a as a soft, but tractable, glassy white foam; yield 529

mg (76%); mp softens to a syrup gradually above

ꢀ92 °C. MS: m/z 1207 [M+H]⁺. IR: 1735cm⁻¹(ester

CꢁO), 1330, 1317, 1144 (SO₂NH). UV (EtOH): 0.1 N

HCl 240 (66.5), 276 (sh), 285 (sh); pH 7 buffer 241

(64.1), 276 (sh), 285 (sh); 0.1 N NaOH 233 (67.7), 276

1

(9.2), 284 (sh). H NMR (CDCl₃): l 0.98 (t, 6 H, CH₃),

1.47 (m, 4 H, CH₃CH₂ꢀ), 1.78 (m, 4 H, CH₃CH₂

CH₂ꢀ), 2.59, 2.90 (2m, 4 H, J_5,63.8, J_6,6%14.7, J_6,NH6.4,

J_5,6%2.6, J_6%,NH7.1 Hz, 6-CH₂), 2.97 (m, 4 H, CH₂-

SO₂NH), 3.93 (2m, 2 H, J_4,59.3, J_5,62.6, J_5,6%3.8 Hz,

H-5), 4.70 (app. t, 2 H, SO₂NH), 5.33 (dd, 2 H, J_1,23.9,

J_2,310.3 Hz, H-2), 5.40 (app. t, 2 H, J_3,49.5 Hz, H-4),

5.70 (d, 2 H, H-1), 6.25 (app. t, 2 H, J_3,49.5 Hz, H-3),

7.33, 7.43, 7.59, 7.82, 7.91, 8.07 (complex m, 30 H, Bz).

Anal. Calcd for C₆₂H₆₄N₂O₁₉S₂: C, 61.78; H, 5.35; N,

2.32. Found: C, 61.50; H, 5.51; N, 2.47.

1

(amide). H NMR (Me₂SO-d₆): l 3.03, 3.21 (2m, 4 H,

J_6,NH4.6, J_5,62.9, J_6,6%14.3, J_6%,

6.1, J_5,6%4.3 Hz,

NH

6-CH₂), 3.76 (m, 2 H, J_4,4-OH5.0, J_3,49.2, J_4,510.0 Hz,

H-4), 3.85 (m, 2 H, H-5), 5.28 (dd, 2 H, J_1,23.7, J_2,3

10.3 Hz, H-2), 5.51 (d, 2 H, H-1), 5.67 (d, 2 H, 4-OH),

5.79 (app. t, 2 H, H-3), 7.47, 7.51, 7.78, 7.95 (complex

m, 30 H, Bz), 8.18 (t, 2H, BzNHꢀ). Strong water peak

noted. Anal. Calcd for C₅₄H₄₈N₂O₁₅·0.9 H₂O: C, 66.10;

H, 5.12; N, 2.86. Found: C, 66.12; H, 5.13; N, 2.89.

The other identiﬁed products obtained from the

column were 8 (ꢀ3%) and the 6-benzamido-6%-amino

compound (1.48 g, 31%) formed by O⁴to N⁶Bz

migration in one ring, but not both. Spectral analysis of

this transient intermediate shortly after it was obtained

from the column gave a FAB mass spectrum identical

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis-

[(1-octylsulfonyl)amino]-h,h-trehalose (10b).—Using the

following quantities of reagents, 10b was obtained by

the same method given in detail for 10a above: 8 (717

mg, 0.74 mmol), NaHCO₃(125 mg, 1.49 mmol), MeOH

(10 mL), Et₃N (202 mg, 2.0 mmol), 1-octanesulfonyl

chloride (319 mg, 1.50 mmol), CH₂Cl₂for reaction

solvent (20 mL); reaction time was 48 h. Column

chromatography on silica gel with (99:1) CHCl₃–

MeOH as eluant gave 10b as a white glassy foam; yield

536 mg (55%); mp softens to a syrup above ꢀ70 °C.

MS: m/z (with LiCl) 1323 [M+Li]⁺, 650 (1/2[M−

16]⁺), 528 [650−PhCOOH]⁺, 406 [528−PhCOOH]⁺.

UV (EtOH; turbid at pH 1 and 7): 0.1 N HCl 242, 276

(sh), 284 (sh); pH 7 buffer 242, 276 (sh), 284 (sh); 0.1 N

NaOH 232 (69.5), 275 (7.4), 284 (sh). ¹H NMR

(CDCl₃): l 0.89 (t, 6 H, CH₃), 1.31, 1.42, 1.78, 2.95

(4m, 16 H, 4 H, 4 H, 4 H, aliphatic CH₂), 2.60, 2.88

(2m, 4 H, 6-CH₂), 3.93 (2m, 2 H, J_4,510.0 Hz, H-5),

4.68 (t, 2 H, SO₂NH), 5.31, 5.34 (dd, 2 H, J_1,23.9, J_2,3

10.3 Hz, H-2), 5.39 (app. t, 2 H, J 9.6, J 9.7 Hz, H-4),

5.71 (d, 2 H, H-1), 6.24 (app. t, 2 H, H-3), 7.32, 7.43,

7.58, 7.83, 7.91, 8.07 (complex m, 30 H, Bz). Anal.

Calcd for C₇₀H₈₀N₂O₁₉S₂: C, 63.81; H, 6.12; N, 2.13.

Found: C, 63.91; H, 6.18; N, 2.33.

1

to 9, as expected, but the H NMR was more compli-

1

cated because of the unsymmetrical molecule. H NMR

(Me₂SO-d₆): l 2.14, 2.31 (2m, 2 H, 6%-H₂NCH₂ꢀ),

ꢀ3.32 (m, water superimposed on 6-BzNHCH₂ꢀ), 3.82

(complex m, 3 H, H-4, H-5, H-5%), 5.33 (dd, 1 H, J_1,2

3.7, J_2,310.5 Hz, H-2), 5.51 (m, 1 H, H-2%), 5.54 (d, 1 H,

H-4%), 5.60 (d, 1 H, H-1), 5.66 (d, 1 H, J_1%,2%3.7 Hz,

H-1%), 5.74 (d, 1 H, J_4,4-OH4.8 Hz, 4-OH), 5.87 (app. t,

1 H, H-3), 6.05 (app. t, 1 H, H-3%), 7.36–8.08 (complex

m, 30 H, Bz), 8.27 (t, 1 H, 6-BzNHCH₂). On a TLC

plate with (97:3) CHCl₃–MeOH as solvent, these com-

ponents had the following R_fvalues: 5 (0.93), 9 (0.91),

6-benzamido-6%-amino intermediate (0.55), 8 (0.13).

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-bis[(1-butylsul-

fonyl)amino]-6,6%-dideoxy-h,h-trehalose (10a).—Solid

NaHCO₃(97 mg, 1.16 mmol) was added to a solution

of the amine dihydrochloride 8 (600 mg, 0.58 mmol) in

MeOH (10 mL). After stirring for 5 min, CH₂Cl₂(50

mL) was added, and the mixture was evaporated at rt.

Dry CH₂Cl₂(2×25 mL) was added and evaporated to

aid complete removal of MeOH. A solution of the

residue in dry CH₂Cl₂(25 mL) was treated with Et₃N

(304 mg, 3.0 mmol) followed by 1-butanesulfonyl chlo-

ride (219 mg 1.40 mmol) and stirred at rt under N₂for

3 days. Volatiles were evaporated; a solution of the

residue in CHCl₃was washed by shaking with water,

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis-

[(1-hexadecylsulfonyl)amino]-h,h-trehalose (10c).—The

crude product obtained by reaction of 8 (500 mg, 0.52

mmol) and 1-hexadecanesulfonyl chloride (390 mg, 1.2

mmol) by the method of 10a, above, was chro-

matographed on silica gel with (99:1) CHCl₃–MeOH as

eluant to give 10c (545 mg, 68%) as a glassy foam. A

second column on a 360 mg portion of this to remove

a trace impurity followed by drying for 24 h gave the

analytical sample; 201 mg; mp softens to a syrup above

114

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

ꢀ45 °C. MS (with LiCl): m/z 1547 [M+Li]⁺. UV

(EtOH, turbid at pH 1 and 7): 0.1 N HCl 246, 277 (sh),

285 (sh); pH 7 buffer 244, 277 (sh), 285 (sh); 0.1 N

NaOH 232 (69.2), 275 (8.15), 284 (sh). ¹H NMR

(CDCl₃): l 0. 88 (t, 6 H, CH₃), 1.24, 1.42, 1.78, 2.96

(4m, 48 H, 4 H, 4 H, 4 H, aliphatic CH₂), 2.60, 2.88

(2m, 4 H, J_5,63.5, J_6,6%14.4, J_6,NH6.5, J_5,6%2.5, J_6%,NH6.5

Hz, 6-CH₂), 3.93 (2m, 2 H, J_4,510.0 Hz, H-5), 4.69

(app. t, 2 H, SO₂NH), 5.32, 5.35 (2d, 2 H, J_1,23.8, J_2,3

10.2 Hz, H-2), 5.40 (app. t, 2 H, H-4, J_3,49.8, J_4,510.0

Hz, H-4), 5.71 (d, 2 H, H-1), 6.25 (app. t, 2 H, H-3),

7.32, 7.43, 7.59, 7.84, 7.92, 8.08 (complex m, 30 H, Bz).

Anal. Calcd for C₈₆H₁₁₂N₂O₁₉S₂: C, 66.99; H, 7.32; N,

1.82; S, 4.16. Found: C, 67.23; H, 7.40; N, 1.75; S, 4.02.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-

2-OH), 4.88 (d, 2 H, J 4.8 Hz, 3-OH), 4.93 (app. d, 4 H,

H-1 and 4-OH), 6.80 (app. t, 2 H, SO₂NH). Anal.

Calcd for C₂₀H₄₀N₂O₁₃S₂: C, 41.37; H, 6.94; N, 4.82.

Found: C, 41.16; H, 7.19; N, 4.62.

6,6% - Dideoxy - 6,6% - bis - [(1 - octylsulfonyl)amino] - h,h-

trehalose (11b).—Compound 10b (469 mg, 0.36 mmol)

was debenzoylated by the procedure given in detail for

11a The product was recrystallized from 2:1 water–

EtOH (30 mL), collected, washed on the funnel with

water and Et₂O, and dried at 82 °C for 20 h; yield 204

mg (82%), mp 187–188 °C with prior sintering. MS

1

(with LiCl): m/z 699 [M+Li]⁺. H NMR (Me₂SO-d₆):

l 0.86 (t, 6 H, CH₃), 1.26, 1.34, 1.64 (3m, 24 H,

aliphatic CH₂), 2.99 (complex m, 8 H, CH₂SO₂NH+

H-4+6-CH), 3.26, 3.32 (m, 4 H, J_1,23.6, J_2,2-OH5.8,

J_2,39.6 Hz, 6-CH%+H-2), 3.55 (m, 2 H, J_3,3-OH4.8 Hz,

H-3), 3.74 (m, 2 H, J_4,59.8, J_5,61.8, J_5,6%6.7 Hz, H-5),

4.70 (d, 2 H, J_2,2-OH5.8 Hz, 2-OH), 4.86 (d, 2 H,

3-OH), 4.92 (m, 4 H, J_4,4-OH5.2 Hz, H-1+4-OH), 6.77

bis[(phenylsulfonyl)amino]-h,h-trehalose (10d).—Com-

pound 8 (1.00 g, 1.05 mmol) was reacted with

benzenesulfonyl chloride (389 mg, 1.05 mmol) by the

procedure given for 10a. Column chromatography on

silica gel with CHCl₃as eluant followed by drying for

20 h gave 10d as a brittle white foam; yield 1.10 g (84%)

mp softens gradually above ꢀ110 °C. MS: m/z 1245

[M+H]⁺, with LiCl 1251 [M+Li]⁺. UV (EtOH, tur-

bid at pH 1and 7): 0.1 N HCl 234, 273 (sh), 284 (sh);

pH 7 buffer 234, 273 (sh), 284 (sh); 0.1 N NaOH 231

(app. t,

2

H, SO₂NH). Anal. Calcd for

C₂₈H₅₆N₂O₁₃S₂·0.5 H₂O: C, 47.91; H, 8.18; N, 3.99.

Found: C, 48.04; H, 8.28; N, 3.97.

6,6% - Dideoxy - 6,6% - bis[(1 - hexadecylsulfonyl)amino] -

h,h-trehalose (11c).—Compound 10c (774 mg, 0.50

mmol) was debenzoylated by the method of 11a, above,

except that a white solid deposited after ꢀ15 min in

the alkaline MeOH solution. The collected solid was

washed with Et₂O (20 mL), suspended in 1:1 MeOH–

water (20 mL), and adjusted to pH 6.5 with HCl. The

solid was collected (350 mg, 76%) and recrystallized

from 9:1 MeOH–water (20 mL) to give a homogeneous

(TLC) ﬁrst crop (172 mg, 37%, mp 185–186 °C) and a

lower-melting second crop (101 mg, 22%). After drying

at 82 °C for 20 h, the ﬁrst crop was used as the

analytical sample. MS (with LiCl): m/z 923 [M+Li]⁺.

¹H NMR (Me₂SO-d₆): l 0.86 (t, 6 H, CH₃), 1.24, 1.34,

1.63 (4m, 24 H, 4 H, 4 H, aliphatic CH₂), 2.99 (complex

m, 8 H, CH₂SO₂+H-4+6-CH), 3.26, 3.32 (complex

m, 4 H, J_1,23.6, J_2,39.8, J_2,2-OH4.8 Hz, H-2 overlapped

by 6-CH%), 3.55 (m, 2 H, J_3,3-OH4.8, J_3,48.5 Hz, H-3),

3.73 (m, 2 H, H-5), 4.71(d, 2 H, J 5.8 Hz, OH-2), 4.86

(d, 2 H, 3-OH), 4.92 (app. d, 4 H, J_1,23.6, J_4,4-OH4.9

Hz, H-1+4-OH), 6.78 (app. t, 2 H, SO₂NHCH₂).

Anal. Calcd for C₄₄H₈₈N₂O₁₃S₂·0.2 H₂O: C, 57.39; H,

9.68; N, 3.04. Found: C, 57.35; H, 10.02; N, 3.01.

6,6% - Dideoxy - 6,6% - bis[(phenylsulfonyl)amino] - h,h-

trehalose (11d).—Compound 10d (1.02 g, 0.82 mmol)

was debenzoylated by the procedure for 11a. The crude

product (500 mg, 98%) was recrystallized from a mix-

ture of boiling EtOH (25 mL) and water (3 mL). The

solid was collected, washed with EtOH, and dried at

82 °C for 20 h; yield 444 mg (86%); mp 217–218 °C.

MS (with LiCl): m/z 627 [M+Li]⁺. UV (EtOH): 0.1 N

HCl 220 (19.0), 264 (1.77), 271 (1.46); pH 7 buffer 220

1

(69.7) 273 (8.46), 284 (sh). H NMR (CDCl₃): l 2.48,

2.75 (2m, 4 H, J_5,64.5, J_6,6%14.4, J_6,NH6.1, J_5,6%2.5,

J_6%,NH7.3 Hz, 6-CH₂), 3.83 (2m, 2 H, H-5), 4.97 (app. t,

2 H, SO₂NH), 5.07 (dd, 2 H, J_1,23.7, J_2,310.3 Hz,

H-2,), 5.26 (dd, 2 H, J_3,49.5, J_4,510.2 Hz, H-4), 5.31 (d,

2 H, H-1), 6.11 (app. t, 2 H, H-3), 7.4, 7.77, 7.88, 8.03

(complex m, 35 H, Bz and Ph). Anal. Calcd for

C₆₆H₅₆N₂O₁₉S₂: C, 63.66; H, 4.53; N, 2.25. Found: C,

63.54; H, 4.50; N, 1.99.

6,6% - Bis[(1 - butylsulfonyl)amino] - 6,6% - dideoxy - h,h-

trehalose (11a).—A solution of NaOMe in MeOH (0.21

mL of 1.74 N, 0.36 mmol) was added to a solution of

10a (430 mg, 0.36 mmol) in dry MeOH (10 mL), and

the solution was allowed to stand under N₂for 1 h. The

solution was diluted with an equal volume of water and

adjusted to pH 5 by portionwise addition of Dowex

50W-X8 (H⁺) cation exchange resin. The mixture was

ﬁltered, and the ﬁltrate was evaporated to dryness. The

residue was washed by trituration and ﬁltration with

Et₂O to extract methyl benzoate. The solid ﬁlter cake

(198 mg) was recrystallized from isopropyl alcohol (20

mL); the white solid was collected by ﬁltration, washed

with 2-PrOH, and dried at 65 °C for 20 h; yield 163 mg

(79%); mp 169–170 °C. MS: m/z 581 [M+H]⁺, 563

[M–OH]⁺, 282 (1/2[M−16]⁺), 1161 [2M+H]⁺. ¹H

NMR (Me₂SO-d₆): l 0.89 (t, 6 H, CH₃), 1.38, 1.64 (2m,

4 each, aliphatic CH₂), 3.03 (m, 8 H, CH₂SO₂NH

overlapped by 6-CH₂), 3.28 (m, 2 H, J_1,23.6, J_2,39.8,

J_2,2-OH5.7 Hz, H-2), 3.31 (m, 2 H, H-4 overlapped by

water), 3.55 (m, 2 H, J_3,410.0) Hz, H-3, 3.75 (m, 2 H,

J_5,62.2, J_5,6%6.5 Hz, H-5), 4.73 (d, 2 H, J 5.7 Hz,

1

(18.5), 264 (1.56), 272 (1.28); 0.1 N NaOH 236 (sh). H

NMR (Me₂SO-d₆): l 2.77 (m, 2 H, J_5,67.3, J_6,6%12.8,

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

115

J_6,NH5.0 Hz, 6-CH), 2.96 (m, 2 H, J_3,48.5, J_4,4-OH5.3,

J_4,510.0 Hz, H-4), 3.07 (m, 2 H, J_5,6%1.4 Hz, 6-CH%,),

3.21 (m, 2 H, J_1,23.5, J_2,39.7, J_2,2-OH6.0 Hz, H-2), 3.50

(m, 2 H, J_2,39.7, J_3,48.5, J_3,3-OH5.0 Hz, H-3), 3.70 (m,

2 H, J_4,510.0 Hz, H-5), 4.64 (d, 2 H, 2-OH), 4.83 (m, 4

H, 3-OH+H-1), 4.92 (d, 2 H, 4-OH), 7.41 (m, 2 H,

SO₂NHCH₂), 7.61, 7.81 (2m, 10 H, phenyl CH). Anal.

Calcd for C₂₄H₃₂N₂O₁₃S₂·5 H₂O: C, 45.78; H, 5.28; N,

4.45. Found: C, 45.78; H, 5.31; N, 4.48.

gradually above 80 °C. Attempts to induce crystalliza-

tion were unsuccessful. MS: m/z 565 [M+H]⁺, 274

(1/2[M−16])⁺, 142 [CH₂NHC₈H₁₇]⁺. ¹H NMR

(Me₂SO-d₆): l 0.86 (t, 6 H, CH₃), 1.25 (br s, 20 H,

CH₃(CH₂)₅), 1.37 (m, 4 H, CH₃(CH₂)₅CH₂), 2.45 (m, 4

H, CH₂NHCH₂-6), 2.56, 2.75 (2m, 4 H, 6-CH₂), 3.04 (t,

2 H, H-4), 3.24 (dd, 2 H, H-2), 3.32 (br, OH+water),

3.53 (m, 2 H, H-3), 3.76 (complex m, 2 H, H-5),

4.4–4.8 (very br, OH’s) 4.84 (d, 2 H, J 3.8 Hz, H-1).

Anal. Calcd for C₂₈H₅₆N₂O₉: C, 59.55; H, 9.99; N, 4.96.

Found: C, 59.23; H, 10.26; N, 4.77.

6,6% - Dideoxy - 6,6% - bis[(N,N - didodecyl)amino] - h,h-

trehalose (12).—A partial solution of 7 (363 mg, 1.0

mmol) in a mixture of dodecyl iodide (652 mg, 2.2

mmol), anhyd NaHCO₃(210 mg, 2.5 mmol), and dry

DMAc (20 mL) was stirred under N₂at rt for 3 days

without signiﬁcant reaction (TLC). The mixture was

heated to 50 °C for 1 h with rapid formation of yellow

color and numerous product spots. Evaporation of

volatiles under high vacuum gave a crude gum (1.36 g).

Column chromatography on SG with (4:1) CHCl₃–

MeOH as eluant gave a mobile band (ca. 0.5 g) which

was shown by MS to be a complex mixture of products

with three to ﬁve dodecyl groups per molecule. This

was followed by a very polar band of material (ca. 350

mg) whose UV behavior and salt-like characteristics

suggested a mixture of quaternary iodide salts. Careful

chromatography (9:1 C/M) of a 230 mg fraction from

the mobile band gave the bis(didodecylamino) com-

pound 12 as a soft waxy solid; 75 mg; mp: softens

above ꢀ75 °C. This sample was characterized only by

6,6%-Dideoxy-6,6%-bis(1-dodecylamino)-h,h-trehalose

(15b).—Solid n-dodecylamine (14 g, excess) and 4 (1.50

g, 1.37 mmol) were combined and heated in an oil bath

(65 °C) to give a clear melt, which was covered with N₂,

stoppered tightly, and heated at 65 °C for 66 h. The

stopper was replaced with a cold trap with a reservoir

ﬁlled with dry ice. By increasing the bath temperature

to 95 °C with evacuation on the oil pump, most of the

excess amine was collected in the cold trap. A solution

of the residue in CHCl₃(100 mL) was washed by

shaking with dilute base (20 mL of water to which was

added 2.8 mL of 1 N NaOH), followed by water (20

mL). Evaporation of the dried organic layer gave 4.13 g

of beige solid. Column chromatography on SG with

(99:1) CHCl₃–MeOH as eluant gave a mobile band of

N-dodecylbenzamide (2.13 g, 90%). Continued elution

with (15:5:1) CHCl₃–MeOH–concd NH₄OH gave 0.68

g (73%) of impure 15b. A second column on the best of

these fractions (0.42 g) with the 15:5:1 mixture gave the

analytical sample. After drying for 48 h, the clear glass

weighed 140 mg (15%); mp softens ꢀ85 °C. MS: m/z

677 [M+H]⁺, 330 (1/2[M−16])⁺, 198 [CH₂NHC₁₂-

1

MS and H NMR spectra. MS: m/z 1013 [M+H]⁺,

1

498 (1/2[M−16])⁺, 366 [CH₂N(C₁₂H₂₅)₂]⁺. H NMR

(Me₂SO-d₆): l 0.86 (t, 12 H, CH₃), 1.25 (br s, 72 H,

CH₃(CH₂)₉), 1.47 (br s, 8 H, CH₃(CH₂)₉CH₂), 2.99 (m,

12 H, ꢀCH₂NCH₂ꢀ6 plus 6-CH₂), 3.27–3.36 (br m, H-2

and H-4 overlapped by water), 3.58 (m, 2 H, H-3), 4.04

(m, 2 H, H-5), 4.88 (d, 2 H, J 3.5 Hz, H-1), 4.9–5.4

(very broad, OH’s, best seen in integral).

1

H₂₅]⁺. H NMR (Me₂SO-d₆): 0.86 (t, 6 H, CH₃), 1.24

(br s, 36 H, CH₃(CH₂)₉), 1.37 (m, 4 H, CH₃(CH₂)₉-

CH₂), 2.43–2.56 (m, 4 H, CH₂NHCH₂-6), 2.57 (dd, 2

H, J_5,6a6.9, J_6a,6b12.2 Hz, H-6a), 2.77 (dd, 2 H, J_5,6b

3.6 Hz, H-6b), 3.04 (app. t, 2 H, H-4, J_3,48.8, J_4,59.7

Hz, H-4), 3.24 (dd, 2 H, J_1,23.6, J_2,39.5 Hz, H-2), 3.32

(br, water, OH), 3.54 (app. t, 2 H, H-3), 3.77 (m, 2 H,

H-5), 4.4–4.8 (very br, OH’s) 4.85 (d, 2 H, H-1). Anal.

Calcd for C₃₆H₇₂N₂O₉·5 CH₃OH: C, 63.26; H, 10.76;

N, 4.04. Found: C, 63.10; H, 11.16; N, 4.03.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis-

[[N-methyl-N-(1-octadecyl)]amino]-h,h-trehalose (14c).

—A solution of 4 (1.00 g, 0.92 mmol) and N-methyloc-

tadecylamine (2.85 g, 10.1 mmol) in acetone (50 mL)

was reﬂuxed under an N₂atmosphere for 66 h with

minimal reaction (TLC). The acetone was evaporated

and replaced with xylenes to which was added NaI (50

mg), and the solution was reﬂuxed for 16 h under N₂.

The cooled solution was evaporated and then evacuated

under a cold trap (see 14b) using a 130 °C oil bath to

remove most of the excess amine. A solution of the

residue (2.37 g) in CHCl₃(100 mL) was washed by

shaking with satd aq NaHCO₃and water (100 mL

Compounds 15a and 15b were obtained directly from

4 without isolation of intermediates 14a and 14b.

6,6% - Dideoxy - 6,6% - bis(1 - octylamino) - h,h - trehalose

(15a).—A solution of 4 (2.00 g, 1.83 mmol) and excess

n-octylamine (4.69 g, 36.3 mmol) in MeOH (60 mL)

was reﬂuxed with protection from moisture for 66 h.

Solvent and excess amine were removed in vacuo. A

solution of the residual gum in CHCl₃(100 mL) was

washed by shaking with water (20 mL) to which was

added 3.7 mL of 1 N NaOH, followed by water.

Evaporation of the dried (Na₂SO₄) organic layer gave a

dark gum (1.17 g), which was applied to a SG column.

Elution ﬁrst with (99:1) CHCl₃–MeOH gave a mobile

band containing N-octylbenzamide; elution with (15:

5:1) CHCl₃–MeOH–concd NH₄OH gave fractions (to-

tal 303 mg) containing impure 15a. Careful chromatog-

raphy of the best fractions (230 mg) using the 15:5:1

mixture gave the analytical sample; after drying for 48

h, the yield of clear glass was 214 mg (21%); mp softens

116

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

each). The dried (Na₂SO₄) organic layer was evapo-

rated to give 2.15 g of dark gum. Column chromatogra-

phy on SG with (49:1) CHCl₃–MeOH gave 0.84 g

(61%) of 14c with minor impurities. After conﬁrmation

of identity by mass spectral analysis (m/z 1498 [M+

H]⁺), this material was used in the deprotection step

without further treatment. See 15c below.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis-

[N,N-bis(2-ethylhexyl)amino]-h,h-trehalose (14d).—A

solution of 4 (1.00 g, 0.92 mmol) and bis(2-ethyl-

hexyl)amine (2.43 g, 10.1 mmol) in xylenes (25 mL), to

which was added NaI (50 mg), was heated for 66 h

150 °C under an N₂atmosphere. The bath was cooled

to 125 °C, and the solvent and excess amine were

distilled off using a trap cooled in dry ice. Column

chromatography of the residue (2.06 g) on SG with

CHCl₃as eluant gave 0.90 g (70%) of 14d with minor

impurities. MS: m/z 1413 [M+H]⁺. This material was

used in the deprotection step without further treatment.

See 15d below.

6,6%-Dideoxy-6,6%-bis[[N-methyl-N-(1-octadecyl)]-

amino]-h,h-trehalose (15c).—A suspension of 14c (790

mg, 0.53 mmol) in MeOH (100 mL) saturated with

NH₃at 0 °C was heated at 60 °C for 18 h in a glass-

lined stainless steel bomb. After cooling, the suspension

was decanted into a ﬁlter funnel without disturbing

some tarry matter stuck on the glass. The solid ﬁlter

cake was crude 15c; 296 mg (64%). The main compo-

nent of the ﬁltrate was benzamide as shown by TLC

comparison with an authentic sample. The crude

product was chromatgraphed on SG with (85:15:1)

CHCl₃–MeOH–concd NH₄OH as eluant to give 178

mg (39%) of puriﬁed 15c. Recrystallization from

MeOH gave the analytical sample; 90 mg; mp softens

gradually above ꢀ98 °C. MS: m/z 873 [M+H]⁺, 428

(1/2[M−16])⁺, 296 [CH₂N(CH₃)C₁₈H₃₇]⁺. ¹H NMR

(Me₂SO-d₆): l 0.86 (t, 6 H, terminal CH₃), 1.25 (br s, 60

H, CH₃(CH₂)₁₅), 1.38 (m, 4 H, CH₃(CH₂)₁₅CH₂), 2.19

(s, 6 H, N(CH₃)), 2.35 (m, 4 H, CH₂N(CH₃)), 2.39 (dd,

2 H, J_5,6a6.7, J_6a,6b13.2 Hz, H-6a), 2.65 (dd, 2 H, J_5,6b

3.9, Hz, H-6b), 3.00 (dd, 2 H, J_3,48.9, J_4,59.5 Hz, H-4),

3.26 (br dd, 2 H, J_1,23.7, J_2,39.3 Hz, H-2), 3.59 (3d, 2

H, J_3,3-OH4.5 Hz, H-3), 3.88 (3d, 2 H, H-5), 4.31 (br, 2

H, 4-OH), 4.45 (d, 2 H, 3-OH), 4.82 (br, 2 H, 2-OH),

4.86 (d, 2 H, H-1). Anal. Calcd for C₅₀H₁₀₀N₂O₉: C,

68.76; H, 11.54; N, 3.21. Found: C, 68.88; H, 11.54; N,

3.28.

(Na₂SO₄), ﬁltered, and evaporated to give 0.59 g of

dark gum containing trehalose products and methyl

benzoate. Column chromatography on SG with (16:4:1)

CHCl₃–MeOH–concd NH₄OH gave 184 mg (37%) of

impure 15d. Chromatography of the best fraction (97

mg) using (9:1) CHCl₃–MeOH gave the analytical sam-

ple as a waxy solid, which was dried at 65 °C for 18 h;

yield 77 mg (15%); mp softens gradually above ꢀ

140 °C. MS: m/z 789 [M+H]⁺, 689 [M−(2-ethyl-

1

hexyl)]⁺, 386 (1/2[M−16])⁺. H NMR (Me₂SO-d₆): l

0.79 (t, 12 H, CH₃of 2-Et), 0.86 (t, 12 H, CH₃of

hexyl), 1.10–1.47 (complex m, 36 H, alkyl CH₂), 2.11–

2.34 (m, 8 H, CH₂NCH₂-6), 2.45, 2.73 (2m, 4 H,

6-CH₂), 2.91 (m, 2 H, H-4), 3.21 (m, 2 H, H-2), 3.59

(m, 2 H, H-3), 3.87 (m, 2 H, H-5), 4.39 (m, 2 H, 2-OH),

4.73 (m, 4 H, 4-OH+3-OH), 4.91 (m, 2 H, H-1). Anal.

Calcd for C₄₄H₈₈N₂O₉: C, 66.97; H, 11.24; N, 3.55.

Found: C, 66.73; H, 11.46; N, 3.76.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis(1-

hexylamino)-h,h-trehalose (17a).—The ditriﬂate inter-

mediate 16 was obtained by a published procedure.³⁴

Under an N₂atmosphere, a solution of 16 (4.00 g, 3.25

mmol) and n-hexylamine (1.32 g, 13.0 mmol) in dry

CH₂Cl₂(50 mL) was stirred at rt for 48 h and then

evaporated. The residual gum was triturated thor-

oughly with water (200 mL), and the solid was collected

by ﬁltration, washed with water, and dried. Column

chromatography of the crude product (5.37 g) on SG

with (49:2) CHCl₃–MeOH as eluant gave 2.62 g (71%)

of 17a suitable for use as an intermediate. A large

homogeneous (TLC) fraction was dried at rt for 24 h

for use as the analytical sample; 1.36 g; mp foam sinters

above 40 °C. MS: m/z 1133 [M+H]⁺, 558 (1/2[M−

16])⁺, 436 [558−PhCOOH]⁺, 105 [Bz]⁺. ¹H NMR

(CDCl₃): l 0.86 (t, 6 H, CH₃), 1.22 (complex m, 16 H,

CH₃(CH₂)₄), 2.31 (m, 8 H, CH₃(CH₂)₄CH₂+6-CH₂),

4.01 (m, 2 H, J_4,510.2, J_5,64.4 Hz, H-5), 5.45 (dd, 2 H,

J_1,23.8, J_2,310.2 Hz, H-2), 5.54 (app. t, 2 H, J_3,49.6 Hz,

H-4), 5.67 (d, 2 H, H-1), 6.23 (app. t, 2 H, H-3),

7.26–7.49, 7.55, 7.79, 7.91, 8.13 (m, 30 H, Bz). 6-NH

absorption was very broad and was best seen in the

integral in the 1.3–1.9 region. Anal. Calcd for

C₆₆H₇₂N₂O₁₅: C, 69.95; H, 6.40; N, 2.47. Found: C,

69.98; H, 6.52; N, 2.47.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-bis(1-decylamino)-

6,6%-dideoxy-h,h-trehalose (17b).—This product was

obtained from 16 and n-decylamine by the same proce-

dure as 17a. Column chromatography (99:1 C/M) gave

1.98 g (49%) of 17b suitable for use as an intermediate;

however, a second column was required to obtain a

homogeneous sample for analysis; 0.98 g (24%); mp

low-melting soft glass sinters B40 °C. MS: m/z 1245

[M+H]⁺, 614 (1/2[M−16])⁺, 492 [614−PhCOOH]⁺,

6,6%-Dideoxy-6,6%-bis[N,N-bis(2-ethylhexyl)amino]-

h,h-trehalose (15d).—A solution of 14d (892 mg, 0.63

mmol) in dry MeOH (15 mL) was treated with a

solution of NaOCH₃in MeOH (3 mL of 1.2 N), and

the solution was stirred under N₂at rt for 4 h. Solvent

was evaporated; a solution of the residue in a large

volume of CHCl₃(300 mL) was shaken with water (50

mL), and the mixture was allowed to stand until the

emulsion separated. The turbid organic layer was dried

1

105 [Bz]⁺. H NMR (CDCl₃): l 0.89 (t, 6 H, CH₃),

0.98–1.39 (m, 32 H, CH₃(CH₂)₈), 1.4–1.9 (very br,

NH), 2.32 (complex m, 8 H, CH₃(CH₂)₈CH₂+6-CH₂),

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

117

attempt to remove the Bz group, if possible, a solution

of the gum in liquid NH₃(50 mL) contained in a

glass-lined bomb equipped with a gas-inlet valve was

allowed to stand at rt for 24 h, and then the NH₃was

allowed to evaporate overnight. Removal of the ben-

zoyl group was not complete, but the byproduct spot

was considerably diminished (TLC). Column chro-

matography on SG with (15:5:1) CHCl₃–MeOH–

4.02 (m, 2 H, H-5), 5.46 (dd, 2 H, H-2), 5.54 (t, 2 H,

H-4), 5.68 (d, 2 H, H-1), 6.24 (t, 2 H, H-3), 7.28–7.60,

7.55, 7.79, 7.91, 8.23 (m, 30 H, Bz). Anal. Calcd for

C₇₄H₈₈N₂O₁₅: C, 71.36; H, 7.12; N, 2.25. Found: C,

71.28; H, 7.35; N, 2.20.

2,3,4,2%,3%,4%-Hexa-O-benzoyl-6,6%-dideoxy-6,6%-bis-

[(2-ethylhexyl)amino]-h,h-trehalose (17c).—This pro-

duct was obtained from 16 (4.53 g) and (9)2-ethyl-

hexylamine (1.05 g) by the method for 17a. Column

chromatography on SG with (99.5:0.5) CHCl₃–MeOH

gave 2.74 g (63%) of 17c suitable for use as an interme-

diate. The largest homogeneous fraction was dried at rt

for 24 h; 1.67 g (38%); mp 56–58 °C with prior sinter-

ing. MS: m/z 1189 [M+H]⁺, 1089 [M−CH₃-

(CH₂)₃CH(Et)]⁺, 586 (1/2[M−16])⁺, 464 [586−Ph-

concd NH₄OH gave 0.55

g of mixed 18b and

monobenzoyl byproduct, followed by 0.62 g of 18b with

a barely detectable trace of sodium (or ammonium)

benzoate. The latter was removed by shaking a CHCl₃

solution with satd NaHCO₃solution and with water.

The glassy crust recovered from the CHCl₃layer was

crystallized by extraction into ꢀ100 mL of boiling

acetone in a Soxhlet apparatus. The waxy solid was

dried at 82 °C for 24 h; 296 mg; mp sinters 115 °C. MS:

m/z 621 [M+H]⁺, 302 (1/2[M−16])⁺, 170 [C₁₀H₂₁-

1

COOH]⁺, 105 [Bz]⁺. H NMR (CDCl₃): l 0.77 (m, 6

H, CH₃of Et), 0.87 (m, 6 H, CH₃of hexyl), 1.19

(complex m, 16 H, alkyl CH), 1.3–1.8 (very br, NH),

2.16, 2.32 (2m, 8 H, CH₂NHCH₂superimposed on

6-CH₂), 4.02 (m, 2 H, H-5), 5.46 (dd, 2 H, H-2), 5.57 (t,

2 H, H-4), 5.67 (d, 2 H, H-1), 6.22 (t, 2 H, H-3),

7.29–7.50, 7.54, 7.77, 7.81, 8.14 (m, 30 H, Bz). Anal.

Calcd for C₇₀H₈₀N₂O₁₅: C, 70.69; H, 6.78; N, 2.36.

Found: C, 70.68; H, 6.95; N, 2.34.

6,6% - Dideoxy - 6,6% - bis(1 - hexylamino) - h,h - trehalose

(18a).—A solution of 17a (2.29 g, 2.02 mmol) in 100

mL of MeOH saturated with NH₃at 0 °C was heated

at 65 °C for 20 h in a glass-lined stainless steel bomb.

The crude product (2.6 g) was chromatographed on SG

with (14:6:1) CHCl₃–MeOH–concd NH₄OH to give

18a; yield of clear brittle glass after drying for 6 h at

65 °C was 0.77 g (75%); mp softens 95 °C. MS: m/z 508

[M+H]⁺, 246 (1/2[M−16])⁺, 114 [C₆H₁₃NHCH₂]⁺.

¹H NMR (Me₂SO-d₆): l 0.86 (t, 6 H, CH₃), 1.25(m, 12

H, CH₃(CH₂)₃), 1.37 (m, 4 H, CH₃(CH₂)₃CH₂), 2.52

(complex m, 6 H, CH₂NH superimposed on 6-CH),

2.76 (dd, 2 H, 6-CH), 3.05 (t, 2 H, H-4), 3.24 (dd, 2 H,

H-2), 3.54 (t, 2 H, H-3), 3.77 (m, 2 H, H-5), 4.4–5.1

(very br, OH’s), 4.78 (br s, 2 H, 3-OH), 4.83 (d, 2 H,

H-1). Anal. Calcd for C₂₄H₄₈N₂O₉: C, 56.67; H, 9.51;

N, 5.51. Found: C, 56.62; H, 9.75; N, 5.51.

1

NHCH₂]⁺. H NMR (Me₂SO-d₆): l 0.86 (t, 6 H, CH₃),

1.25 (br s, 28 H, CH₃(CH₂)₇), 1.37 (m, 4 H, CH₃-

(CH₂)₇CH₂), 2.52, 2.75 (2m, 8 H, CH₂NHCH₂and

6-CH₂), 3.05 (t, 2 H, H-4), 3.24 (dd, 2 H, H-2), 3.55 (m,

2 H, H-3), 3.76 (m, 2 H, H-5), 3.9-4.8 (very br, OH’s),

4.84 (m, 2 H, H-1). Anal. Calcd for C₃₂H₆₄N₂O₉: C,

61.91; H, 10.39; N, 4.51. Found: C, 61.84; H, 10.45; N,

4.43.

6,6%-Dideoxy-6,6%-bis[(2-ethylhexyl)amino]-h,h-tre-

halose (18c).—A solution of NaOMe–MeOH (2.6 mL

of 1.2 N) was added to a solution of 17c (1.61 g, 1.35

mmol) in dry MeOH (20 mL). The reaction mixture

was stirred under N₂at rt for 1 h and then treated with

cation exchange resin with subsequent workup as de-

scribed for 18b. Column chromatography of the crude

product (0.86 g) on SG with (80:20:1) CHCl₃–MeOH–

concd NH₄OH gave a rough separation of 18c (282 mg,

37%) from a byproduct shown to be a (mono) N-ben-

zoyl derivative of 18c (see Section 2). A second column

gave the analytical sample as a brittle foam, which was

dried at 65 °C for 30 h; 110 mg (14%); mp softens

ꢀ158 °C. MS: m/z 565 [M+H]⁺, 274 (1/2[M−16])⁺,

1

142 [alkylNHCH₂ꢀ]⁺. H NMR (Me₂SO-d₆): l 0.85 (m,

6,6% - Bis(1 - decylamino) - 6,6% - dideoxy - h,h - trehalose

(18b).—Compound 17b (0.89 g, 0.71 mmol) was com-

bined with some previous column fractions totaling 2.2

g known to contain 17b, but of lesser quality, and a

solution of the composite sample in dry MeOH (85 mL)

was treated with 1.2 N NaOMe–MeOH solution (2.60

mL) and stirred at rt for 1 h. The solution was diluted

with an equal volume of water and neutralized by

portionwise addition of Dowex 50W-X8 (H⁺) resin.

After ﬁltration, the mixture was evaporated and re-

evaporated with EtOH to give 1.92 g of pale yellow

gum. MS analysis showed the desired product 18b, 621

[M+H]⁺, and a signiﬁcant byproduct, 725 [M+H]⁺,

corresponding to 18b with a single benzoyl group. In an

12 H, CH₃of Et and hexyl), 1.10–1.42 (m, 18 H, alkyl

CH), 2.38, 2.47 (2m, 4 H, CH(Et)CH₂NH), 2.59, 2.76

(2dd, 4 H, 6-CH₂), 3.06 (t, 2 H, H-4), 3.24 (dd, 2 H,

H-2), 3.55 (m, 2 H, H-3), 3.78 (m, 2 H, H-5), 4.2–5.2

(very br, OH’s), 4.79 (d, 2 H, 3-OH), 4.86 (d, 2 H, H-1).

Anal. Calcd for C₂₈H₅₆N₂O₉: C, 59.55; H, 9.99; N, 4.96.

Found: C, 59.42; H, 10.17; N, 4.87.

2,3,4,2%,3%,4%,6% - Hepta - O - benzoyl - 6 - O - (triﬂuoro-

methylsulfonyl)-h,h-trehalose (19).—Hepta-O-benzoyl-

trehalose had been obtained by a literature procedure,³⁴

and a 4.10 g sample was converted to 19 in 99% crude

yield³⁴for their compound 39. MS (with LiCl): m/z

1209 [M+Li]⁺, 607, 579 [unsymmetrical glucopyran-

osyl moieties from fragmentation of the glycosidic

118

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

OH+4%-OH, J 4.7, 5.1, 4.7 Hz),^†4.87 and 4.88 (2m, 2

H, H-1 and H-1%). Anal. Calcd for C₂₀H₃₉NO₁₀: C,

52.97; H, 8.67, N, 3.09. Found: C, 52.71; H, 8.55; N,

3.02.

1

bond]⁺, 105 [Bz]⁺. H NMR (CDCl₃): 3.86, 4.00 (2m,

4 H, 6-CH₂), 4.28 (m, 2 H, H-5), 5.49 (m, 3 H, H-2’s

and H-4 of ring with 6-OTf), 5.69 (m, 1 H, H-4 of ring

with 6-OBz), 5.70, 5.77 (2d, 2 H, H-1’s), 7.24–8.15

(m’s, 35 H, Bz). Anal. Calcd for C₆₂H₄₉F₃O₂₀S: C,

61.90; H, 4.10. Found: C, 61.94; H, 3.74.

Intermediates 23a–d were all obtained by a literature

method,³⁶which is illustrated below for 23a.

Hexaneimidic acid, methyl ester, hydrochloride 23a.—

Under an N₂atmosphere, a solution of hexanenitrile

(2.71 g, 27.8 mmol) and dry MeOH (5 mL) in dry Et₂O

(40 mL) was cooled in an ice–water bath, and gaseous

HCl was bubbled in for 15 min. The mixture was stored

in the freezer overnight. Solvents were evaporated, and

the residue was triturated thoroughly with cold Et₂O

(20 mL). The slurry of white crystals was ﬁltered under

N₂pressure, washed with cold Et₂O, and dried in vacuo

over P₂O₅; yield 4.18 g (91%). Used ‘as is’ without

characterization.

2,3,4,2%,3%,4%6%-Hepta-O-benzoyl-6-deoxy-6-[(2-ethyl-

hexyl)amino]-h,h-trehalose (20).—A solution of 19

(4.46 g, 3.71 mmol) and (9)2-ethylhexylamine (1.01 g,

7.8 mmol) in CH₂Cl₂(50 mL) was allowed to stand at

rt for 20 h and then evaporated. The crude product was

chromatographed on SG with CHCl₃as eluant. The

leading and trailing edges of the main product band

(total 4.2 g, 96%) were rejected because of trace con-

taminants. The homogeneous middle cut gave the ana-

lytical sample as a brittle foam after drying at rt for 18

h; 2.29 g (52%); mp sinters ꢀ60 °C. MS: m/z 1182

[M+H]⁺, 586, 579 [unsymmetrical glucopyranose frag-

ments]⁺, 464 [586−BzOH]⁺, 105 [Bz]⁺. ¹H NMR

(CDCl₃): l 0.75(m, 3 H, CH₃of Et), 0.86 (m, 3 H, CH₃

of hexyl), 1.0–1.3 (br m, 9 H, alkyl CH), 1.4–1.8 (very

br, NH), 2.14, 2.27 (2m, 4 H, CH(Et)CH₂NH plus

6-CH₂of ring with alkylamine), 3.92, 4.03 (2m, 3 H,

6-BzOCH₂plus H-5), 4.25 (m, 1 H, H-5 of ring with

6-BzOCH₂), 5.46 (dd, 2 H, H-2’s), 5.57, 5.64 (2d, 2 H,

H-4’s), 5.72 (m, 2 H, H-1’s), 6.26 (m, 2 H, H-3’s),

7.15–8.18 (m’s, 35 H, Bz). UV (EtOH): 0.1 N HCl 233

(80.2), 275 (11.3), 284 (sh); pH 7 buffer 242 (80.3), 276

(sh), 283 (sh); 0.1 N NaOH 238 (88.4), 276 (sh), 283

(sh). Anal. Calcd for C₆₉H₆₇NO₁₇: C, 70.10; H, 5.71; N,

1.18. Found: C, 70.11; H, 5.46; N, 1.16.

Intermediates 23b,c, and d were obtained from the

1

appropriate nitriles by the same method as 23a. An H

NMR spectra was obtained for methyl dodecaneimi-

1

date hydrochloride 23b. H NMR (Me₂SO-d₆): l 0.86

(t, 3 H, dodecyl CH₃), 1.25 (br s, 16 H, CH₃(CH₂)₈),

1.59 (m, 2 H, (CH₂)₈CH₂), 2.62 (t, 2 H, CH₂CꢁNH⁺₂),

4.07 (s, 3 H, OCH₃), 7.18, 7.35, 7.52 (3s, 3 H, J 49.4

Hz, NH).

6,6% - Dideoxy - 6,6% - bis[(1 - iminohexyl)amino] - h,h -

trehalose (24), mixture with 6,6%-dideoxy-6-(1-imino-

hexyl)amino-6%-(1-oxohexyl)amino-h,h-trehalose

(25),

compound with acetic acid [10:1:25].—With protection

from moisture, a partial solution of 7 (732 mg, 2.0

mmol) in dry MeOH (250 mL) was heated to boiling

and then cooled to ꢀ50 °C to increase solubility.

6-Deoxy-6-(2-ethylhexyl)amino-h,h-trehalose (21).—

A solution of NaOMe in MeOH (2.3 mL of 1.2 N) was

added to a suspension of 20 (4.02 g, 3.4 mmol) in dry

MeOH (225 mL), and the mixture was stirred under N₂

for 2 h and then evaporated. The residue was washed

by trituration and decantation with hexane (2×50

mL), and the dried crude product (1.75 g) was chro-

matographed on SG. Elution with (12:8:1) CHCl₃–

MeOH–concd NH₄OH gave 190 mg (10%) of the

N-benzoyl byproduct followed by 21 (1.32 g, 86%).

Recrystallization of the latter from boiling acetone gave

a granular white solid, which was dried at rt for 18 h;

yield 1.13 g (73%); mp sinters ꢀ106 °C. MS: m/z 454

[M+H]⁺. ¹H NMR (Me₂SO-d₆): l 0.84 (m, 6 H, CH₃’s

of Et and hexyl), 1.1–1.4 (br m, 9 H, alkyl CH), 2.38,

2.48 (2m, 2 H, CH(Et)CH₂NH), 2.60 (dd, 1 H, J_5,6a6.9,

J_6a,6b12.6 Hz, 6-CH_a), 2.77 (dd, 1 H, J_5,6b3.5 Hz,

6-CH_b), 3.05 (dd, 1 H, J_3,48.6, J_4,510.0 Hz, H-4), 3.14

(3d, 1 H, J_3%,4%8.9, J_4%,5%9.9, J_4%,4%-OH5.1 Hz, H-4%), 3.24

and 3.25 (2m, 2 H, J_1,2J_1%,2%3.6, J_{2,3 or 2%,3%}9.7, J_{2%,3% or 2,3}

9.6 Hz, H-2 and H-2%), 3.40–3.61 (complex m, 4 H,

6%-CH₂OH+H-3+H-3%), 3.65 (m, 1 H, H-5%), 3.79 (m,

1 H, H-5), 4.36 (t, 1 H, J 5.8 Hz, 6%-CH₂OH)*, 4.62 (d,

1 H, J_2%,2%-OH6.3 Hz, 2%-OH)*, 4.77 (m, 3 H, 2-OH+4-

,

Freshly activated Linde 3 A molecular sieve (6 g) was

added, followed by methyl hexaneimidate hydrochlo-

ride (1.32 g, 8.0 mmol) and dry Amberlite IRA-400

anion exchange resin (2.0 g, OH⁻form, nominal 4

mmol/g exchange capacity). The mixture was stirred at

rt under N₂for 3 days. TLC showed two product spots,

but much unreacted starting materials, so the mixture

was heated in a 50 °C oil bath for 3 days more. The

suspended solids were removed by ﬁltration, and the

ﬁlter cake (resin and sieves) was washed liberally with

MeOH. Evaporation of the ﬁltrate gave a residue that

was washed by trituration and decantation with hexane

(2×25mL) to remove some imidate ester free base.

Flash column chromatography of the dried residue (1.2

g) on SG with (20:20:1) CHCl₃–MeOH–HOAc gave a

mobile band of mixed organic and inorganic material

whose main organic component (28 mg) was 25 by MS:

m/z 536 [M+H]⁺; TLC (20:20:1): R_f0.58. Continued

^†These clearly-deﬁned OH peaks only account for ﬁve of

the seven OH protons; however, the region 4.30–4.82 inte-

grates for seven protons, and very broad absorption (tenta-

tively assumed to be the 3- and 3%-OH’s) is also evident in the

baseline. All absorption in this region disappears upon ex-

change with D₂O.

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

119

elution gave a similar band whose main organic compo-

nent was 24 by MS: m/z 535 [M+H]⁺; TLC (20:20:1):

R_f0.17. A turbid solution of 24 in water (5 mL) was

ﬁltered, and the ﬁltrate was applied to a Bio-Beads™

SM-4 resin column (20–50 mesh, 40 mL packed vol-

ume). Elution with water removed most of the inor-

ganic matter. Elution with (9:1) water–MeOH and

ﬁnally with (1:1) gave 190 mg of 24 and leached resin.

This solid was stirred vigorously into a ﬁne suspension

in Et₂O, ﬁltered under N₂pressure, and the solid was

washed twice with Et₂O and dried for 24 h; yield 111

mg (8% as the composition given below); mp ꢀ125 °C

with prior sintering. MS: m/z 535 [M+H]⁺, 259 (1/

and excess 30 (1.86 g, 5.2 mmol) in dry DMF (120 mL)

was stirred under N₂at rt for 6 days, although the

stench of methyl mercaptan became obvious in a few

hours. Volatiles were evaporated, and the solid residue

was stirred with Et₂O (100 mL) and collected by ﬁltra-

tion. Column chromatography of the crude product

(1.43 g, 75%) with (9:1) CHCl₃–MeOH as eluant gave

a white solid suitable for use as an intermediate; 1.00 g

(52%); mp 128–130 °C. MS: m/z 961 [M+H]⁺corre-

sponding to the desired product with four CBz groups

and a weak 827 [M+H]⁺corresponding to a similar

product with three CBz groups. Since all CBz groups

would be removed in the ﬁnal step, this latter compo-

1

2[M−16])⁺, weak 536 [M+H]⁺of 25. H NMR of 24

nent was of no consequence. H NMR (Me₂SO-d₆): l

(Me₂SO-d₆): l 0.87 (t, 6 H, CH₃), 1.27 (m, 8 H,

CH₃(CH₂)₂), 1.57 (m, 4 H, CH₃(CH₂)₃CH₂), 2.39 (m, 4

H, CH₂CꢁNH), 3.05 (t, 2 H, H-4), 3.26 (m, 4 H, H-2,

superimposed on 6-CH), 3.42 (br s, 2 H, 6-CH), 3.56 (t,

2 H, H-3), 3.93 (m, 2 H, H-5), 4.90 (d, 2 H, H-1), OH

and NH very broad, best seen in integral and baseline.

Very strong singlet at 1.70 (CH₃of HOAc). A number

of small peaks, some barely visible above the baseline,

correspond to those of component 25 of the 28 mg

byproduct band. Anal. Calcd for C₂₄H₄₆N₄O₉·0.1

C₂₄H₄₅N₃O₁₀·2.5 C₂H₄O₂: C, 51.08; H, 8.26; N, 8.16.

Found: C, 51.03; H, 8.42; N, 7.88.

3.03 (m, 2 H, H-4), 3.26 (complex m, 4 H, H-2 superim-

posed on 6-CH), 3.53 (m, 2 H, H-3), 3.80 (m, 2 H,

6-CH%), 3.92 (m, 2 H, H-5), 4.91 (2d, 4 H, 2-OH+3-

OH), 4.96 (d, 2 H, J 3.9 Hz, H-1), 5.03 (s, 4 H, CH₂of

CBz), 5.20 (m, 6 H, CH₂of CBz superimposed on

4-OH), 7.37 (complex m, 20 H, aromatic CH of CBz),

8.54 (m, 2 H, 6-CH₂NH), 11.53 (m, 2 H, CBzNH).

Water noted. Anal. Calcd for C₄₆H₅₂N₆O₁₇·0.1

C₃₈H₄₆N₆O₁₅·2 H₂O: C, 57.12; H, 5.49; N, 8.83. Found:

C, 57.14; H, 5.37, N, 8.81.

6,6%-Bis[(aminoiminomethyl)amino]-6,6%-dideoxy-h,h-

trehalose (6,6%-dideoxy-6,6%-diguanidino-h,h-trehalose)

(32).—A suspension of 31 (880 mg, 0.84 mmol) in

MeOH (100 mL) was warmed until solution was ob-

tained and then allowed to cool without stirring.

6 - Amino - 6,6% - dideoxy - 6% - [(1 - iminododecyl)amino]-

h,h-trehalose (26) and 6,6%-dideoxy-6,6%-bis[(1-iminodo-

,

decyl)amino]-h,h-trehalose (27).—Linde 3 A molecular

38

sieves (6 g) were added to a partial solution of 7 (145

mg, 0.36 mmol) and methyl dodecaneimidate hydro-

chloride 23b (161 mg, 0.7 mmol) in MeOH (140 mL),

and the mixture was stirred at rt for 48 h. The pulver-

ized sieves were ﬁltered off and washed with MeOH;

the ﬁltrate was evaporated to give a solid mixture of at

least six components (TLC). Column chromatography

on 70–230 mesh SG with (10:2:3) n-BuOH–HOAc–

water as eluant gave, after evaporation of solvent and

lyophilization of a water solution of the residues, 14 mg

(ꢀ6%) of 26 identiﬁed only by MS: m/z 522 [M+H]⁺

CHCl₃(1 mL), water (1 mL), and 10% Pd–C (200

mg) were added, and the mixture was hydrogenated at

atmospheric pressure until H₂uptake ceased after

ꢀ1.5 h. The mixture was ﬁltered through a thin Celite

pad under N₂pressure, and the clear colorless ﬁltrate

was evaporated. A solution of the residue in MeOH (25

mL) was treated slowly dropwise with Et₂O (250 mL),

and the deposit of dense white crystalline dihydrochlo-

ride salt was collected by ﬁltration under N₂pressure,

washed with Et₂O, and dried at 100 °C for 24 h; yield

412 mg (98%); mp sinters above ꢀ120 °C then decom-

poses with foaming at 180 °C. MS: m/z 425 [M+H]⁺,

1

and 24 mg (ꢀ9%) of 27. MS: m/z 703 [M+H]⁺. H

1

NMR (D₂O): l 0.78 (t, 6 H, CH₃), 1.19 (br s, 32 H,

CH₃(CH₂)₈), 1.58 (br m, 4 H, CH₃(CH₂)₈CH₂), 1.81(s,

HOAc), 2.45 (br m, 4 H, CH₂C(ꢁNH⁺₂)), 3.23 (app. t, 2

H, J_3,4+J_4,518.8 Hz, H-4), 3.40–3.48 (2m, 4 H, H-5+

H-2), 3.59 (m, 2 H, H-5%), 3.76 (m, 2 H, J_2,3+J_3,418.5

Hz, H-3), 3.89 (m, 2 H, H-4), 5.00 (br s, 2 H, J_1,22.8

Hz, H-1).

204 (1/2[M−16])⁺. H NMR (Me₂SO-d₆): l 3.06 (t, 2

H, H-4), 3.33 (complex m, 6 H, 6-CH₂+H-2), 3.57 (t,

2 H, H-3), 3.92 (m, 2 H, H-5), 5.00 (d, 2 H, J 3.9 Hz,

H-1), 5.7–7.5 (very br, H⁺, NH, OH). Anal. Calcd for

C₁₄H₂₈N₆O₉·2 HCl·0.2 H₂O: C, 33.57; H, 6.12; Cl,

14.15; N, 16.78. Found: C, 33.61; H, 6.00; Cl, 14.02; N,

16.69.

6,6% - Bis[[[benzyloxycarbonylamino - N - benzyloxycar-

bonyl]iminomethyl]amino] - 6,6% - dideoxy - h,h - trehalose

(31).—N,N% - Bis(benzyloxycarbonyl) - S - methyliso-

thiourea (30) was obtained as a clear colorless oil in

99% crude yield from commercial S-methylisothiourea

hemisulfate (10.0 g, Sigma) and benzyl chloroformate

by the method of Tian et al.³⁶MS: m/z 359 [M+H]⁺.

A solution of diaminotrehalose 7 (716 mg, 2.0 mmol)

Growth inhibitory assays.—The MIC was determined

using either a colorimetric microdulition broth assay or

a macrodilution broth assay. The microdilution broth

assay is described elsewhere;^37,38; the macrodilution

broth assay was similar except that the assay volume

was 1 mL and 12×75 mm glass tubes were used

instead of microtiter plates. M. tuberculosis H37Ra was

obtained from the American Type Culture Collection,

120

J.D. Rose et al. / Carbohydrate Research 337 (2002) 105–120

14. Butler, D. Nature 2000, 406, 670–672.

15. Yew, W. W.; Chau, C. H. Drugs Aging 1997, 10, 405–

410.

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were clinical isolates obtained from the National Jewish

Center for Immunology and Respiratory Diseases,

Denver, CO.

16. Barry, C. E., III; Slayden, R. A.; Sampson, A. E.; Lee, R.

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Acknowledgements

This work was supported by the National Institute of

Allergy and Infectious Diseases grants UO1-A131697,

RO1-A138667, and RO1-AI45317. The authors also

gratefully acknowledge the many helpful discussions

with our collaborator, Dr Michael R. McNeil (Colo-

rado State University). The strains of M. a6ium were

kindly provided by Dr Leonid Heifets, National Jewish

Center for Immunology and Respiratory Diseases,

Denver, CO. We also want to acknowledge Dr James

M. Riordan, Mr Marion Kirk, and Ms Christine

Richards for the spectroscopic and elemental analyses.

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Barry, D. C. J. Med. Chem. 1990, 33, 883–894.

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Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis

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